PROJECT SUMMARY The cell division pathway in bacteria is a fundamental and highly conserved physiological pathway that enables a single mother cell to divide into two identical daughter cells by mitosis. This pathway is essential for proliferation and colonization by bacteria. A detailed understanding of this pathway will provide fundamental, molecular knowledge that will improve the future design of new antibiotics and therapeutics targeting this pathway. Here, we propose experiments to uncover biochemical and functional roles of several key cell division proteins. Early in the cell division pathway, a protein structure with ring-like architecture, called the Z-ring, assembles at the site of cell division. Multiple protein systems ensure that assembly of the ring occurs at midcell, adjacent to the membrane and away from the nucleoid. In Escherichia coli, the Min system, which includes MinC, MinD, and MinE, exhibits polar oscillation and antagonizes FtsZ-ring assembly at the cell poles. MinC directly associates with FtsZ to inhibit FtsZ polymerization, thus preventing Z-ring assembly, and forms a complex with MinD, which establishes the cellular location of MinC. As FtsZ polymerization is antagonized at the poles in vivo by MinC, FtsZ polymers coalesce at midcell to form the Z-ring, tethered to the inner leaflet of the cytoplasmic membrane through direct interactions with membrane-associated ATPase FtsA. Once the mature Z-ring assembles in vivo, FtsZ polymerization is antagonized by a cellular network of proteins that modulate FtsZ polymer assembly. To understand the biochemical mechanisms of systems that regulate Z-ring assembly in vitro and in vivo, we will investigate direct interactions between FtsZ and FtsZ-interacting proteins and probe the functional activities of FtsZ-interacting proteins, including FtsA, MinC and ZapE. We are proposing experiments to elucidate the biochemical mechanisms of FtsA, including ATP utilization, oligomerization, phospholipid binding, recruitment of FtsZ, regulation of FtsZ polymerization, and interactions with late stage division proteins. To understand how MinC interacts with FtsZ and forms copolymers with MinD we are incorporating new genetic tools, phenotypic screens and in vitro biochemical assays to map the FtsZ binding site on MinC, probe activation of MinD, and evaluate the role of MinCD polymers on FtsZ assembly in vivo and in vitro. We will also investigate how ZapE, a recently reported cell division ATPase, impacts division and FtsZ in vitro and in vivo and probe ZapE structure. Together, these studies will uncover key mechanistic steps that are essential for cell division and provide insight to advance current models for Z-ring assembly and constriction based on biophysical interactions.